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J Clin Virol. 2015 May;66:95-9. doi: 10.1016/j.jcv.2015.03.015. Epub 2015 Mar 17.

Evaluation of DNA extraction methods for the detection of Cytomegalovirus in dried blood spots.

Author information

1
Newborn Screening and Molecular Biology Branch, Centers for Disease Control and Prevention, 4770 Buford Hwy. NE, Atlanta, GA 30341, USA. Electronic address: duk5@cdc.gov.
2
Newborn Screening and Molecular Biology Branch, Centers for Disease Control and Prevention, 4770 Buford Hwy. NE, Atlanta, GA 30341, USA.
3
Measles, Mumps, Rubella, and Herpesvirus Laboratory Branch, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30329, USA.

Abstract

BACKGROUND:

Dried blood spots (DBS) are collected universally from newborns and may be valuable for the diagnosis of congenital Cytomegalovirus (CMV) infection. The reported analytical sensitivity for DBS testing compared to urine or saliva varies greatly across CMV studies. The purpose of this study was to directly compare the performance of various DNA extraction methods for identification of CMV in DBS including those used most often in CMV studies.

STUDY DESIGN:

Whatman(®) Grade 903 filter paper cards were spotted with blood samples from 25 organ transplant recipients who had confirmed CMV viremia. Six DNA extraction methods were compared for relative yield of viral and cellular DNA: 2 manual solution-based methods (Gentra Puregene, thermal shock), 2 manual silica column-based methods (QIAamp DNA Mini, QIAamp DNA Investigator), and 2 automated methods (M48 MagAttract Mini, QIAcube Investigator). DBS extractions were performed in triplicate followed by real-time quantitative PCR (qPCR).

RESULTS:

For extraction of both viral and cellular DNA, two methods (QIAamp DNA Investigator and thermal shock) consistently gave the highest yields, and two methods (M48 MagAttract Mini and QIAamp DNA Mini) consistently gave the lowest yields. There was an average 3-fold difference in DNA yield between the highest and lowest yield methods.

CONCLUSION:

The choice of DNA extraction method is a major factor in the ability to detect low levels of CMV in DBS and can largely account for the wide range of DBS sensitivities reported in studies to date.

KEYWORDS:

Dried blood spots; Newborn screening; qPCR

PMID:
25866346
PMCID:
PMC4517665
DOI:
10.1016/j.jcv.2015.03.015
[Indexed for MEDLINE]
Free PMC Article

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